The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction. The wild-type SMC1 gene has been cloned. The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD. Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino-terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail. These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes. The SMC1 gene is essential for viability. Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation. The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination. Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality. Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells. This phenotype of the smc1-2 mutant is not RAD9 dependent. Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, it is proposed that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division (Strunnikov, 1993).
A chromosomal protein that plays an essential role in mitotic chromosome condensation in Xenopus egg extracts has been characterized. Two polypeptides, designated XCAP-C and XCAP-E, associate with each other in extracts, presumably forming a heterodimer. During chromosome assembly in mitotic extracts, XCAP-C/E is recruited to the chromatin and forms a discrete internal structure within assembled chromosomes. Antibody blocking experiments show that XCAP-C function is required for both assembly and structural maintenance of mitotic chromosomes in vitro. Deduced amino acid sequences reveal that the two polypeptides share common structural motifs, consisting of an N-terminal NTP-binding domain, two central coiled-coil regions, and a C-terminal conserved domain. These motifs are highly conserved in a protein family, members of which have been identified recently in both prokaryotes and eukaryotes (Harano, 1994).
The cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified, is described. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II (Saitoh, 1994).
Fission yeast temperature-sensitive mutants cut3-477 and cut14-208 fail to condense chromosomes but small portions of the chromosomes can separate along the spindle during mitosis, producing phi-shaped chromosomes. Septation and cell division occur in the absence of normal nuclear division, causing the cut phenotype. Fluorescence in situ hybridization demonstrate that the contraction of the chromosome arm during mitosis is defective. Mutant chromosomes are apparently not rigid enough to be transported poleward by the spindle. Loss of the cut3 protein by gene disruption fails to maintain the nuclear chromatin architecture even in interphase. Both cut3 and cut14 proteins contain a putative nucleoside triphosphate (NTP)-binding domain and belong to the same ubiquitous protein family that includes the budding yeast Smc1 protein. The cut3 mutant is suppressed by an increase in the cut14+ gene dosage. The cut3 protein, having the highest similarity to the mouse protein, is localized in the nucleus throughout the cell cycle. Plasmids carrying the DNA topoisomerase I gene partly suppress the temperature sensitive phenotype of cut3-477, suggesting that the cut3 protein might be involved in chromosome DNA topology (Saka, 1994).
The characterized SMC2 (structural maintenance of chromosomes) gene has been found to encode a new Saccharomyces cerevisiae member of the growing family of SMC proteins. This family of evolutionary conserved proteins was introduced with the identification of SMC1, a gene essential for chromosome segregation in budding yeast. The analysis of the putative structure of the Smc2 protein (Smc2p) suggests that it defines a distinct subgroup within the SMC family. This subgroup includes the ScII, XCAPE, and cut14 proteins characterized concurrently. Smc2p is a nuclear, 135-kD protein that is essential for vegetative growth. The temperature-sensitive mutation, smc2-6, confers a defect in chromosome segregation and causes partial chromosome decondensation in cells arrested in mitosis. The Smc2p molecules are able to form complexes in vivo both with Smc1p and with themselves, suggesting that they can assemble into a multimeric structure. In this study the first evidence is presented that two proteins belonging to two different subgroups within the SMC family carry nonredundant biological functions. Based on genetic, biochemical, and evolutionary data it is proposed that the SMC family is a group of prokaryotic and eukaryotic chromosomal proteins that are likely to be one of the key components in establishing the ordered structure of chromosomes (Strunnikov, 1995).
Purification and characterization are reported of chromosome condensation protein complexes (termed condensins) containing XCAP-C and XCAP-E, two Xenopus members of the SMC family. Sucrose density gradient centrifugation reveals two major forms of condensins. The 8S form is a heterodimer of XCAP-C and XCAP-E, whereas the 13S form contains three additional subunits. One of them is identified as a homolog of the Drosophila Barren protein, whose mutation shows a defect in chromosome segregation. Chromosomal targeting of condensins is mitosis-specific and is independent of topoisomerase IIalpha. 13S condensin is required for condensation, as demonstrated by immunodepletion and rescue experiments. These results suggest that the condensin complexes represent the most abundant structural components of mitotic chromosomes and play a central role in driving chromosome condensation (Hirano, 1997).
13S condensin is a five-subunit protein complex that plays a central role in mitotic chromosome condensation in Xenopus egg extracts. Two core subunits of this complex, XCAP-C and XCAP-E, belong to an emerging family of putative ATPases, the SMC family. 13S condensin has a DNA-stimulated ATPase activity and exhibits a high affinity for structured DNAs such as cruciform DNA. 13S condensin is able to introduce positive supercoils into a closed circular DNA in the presence of bacterial or eukaryotic topoisomerase I. The supercoiling reaction is ATP-dependent. It is proposed that 13S condensin wraps DNA in a right-handed direction by utilizing the energy of ATP hydrolysis. This reaction may represent a key mechanism underlying the compaction of chromatin fibers during mitosis (Kimura, 1997).
Xenopus 13S condensin converts interphase chromatin into mitotic-like chromosomes, and, in the presence of ATP and a type I topoisomerase, introduces + supercoils into DNA. The specific production of + trefoil knots in the presence of condensin and a type II topoisomerase shows that condensin reconfigures DNA by introducing an ordered, global, + writhe. Knotting requires ATP hydrolysis and cell cycle-specific phosphorylation of condensin. Condensin bind preferentially to + supercoiled DNA in the presence of ATP but not in its absence. These results suggest a mechanism for the compaction of chromatin by condensin during mitosis (Kimura, 1999).
Condensin is a multisubunit protein complex that reconfigures DNA structure in an ATP-dependent manner in vitro and plays a central role in mitotic chromosome condensation in Xenopus egg cell-free extracts. The Xenopus 13S condensin complex (13SC) is composed of two subcomplexes: an 8S core subcomplex (8SC) consisting of two structural maintenance of chromosomes (SMC) subunits (XCAP-C and -E) and an 11S regulatory subcomplex (11SR) containing three non-SMC subunits (XCAP-D2, -G, and -H). The biochemical and functional dissection of this chromosome condensation machinery is reported in this study. Although both 8SC and 13SC can bind to DNA in vitro and contain the SMC ATPase subunits, only 13SC is active as a DNA-stimulated ATPase and supports ATP-dependent supercoiling activity. In the cell-free extracts, 13SC is the active form that binds to chromosomes and induces their condensation. Neither 11SR nor 8SC alone is able to bind to chromatin. These results suggest that the non-SMC subunits have dual roles in the regulation of condensin functions: one is to activate SMC ATPases and the other is to allow the holocomplex to associate with chromatin in a mitosis-specific manner (Kimura, 2000).
The condensin complex in frog extracts, containing two SMC and three non-SMC subunits, promotes mitotic chromosome condensation, and its supercoiling activity increases during mitosis by Cdc2 phosphorylation. Fission yeast has the same five-member condensin complex, each of which is essential for mitotic condensation. The condensin complex was purified and the subunits were identified by microsequencing. Cnd1, Cnd2, and Cnd3, three non-SMC subunits showing a high degree of sequence conservation to frog subunits, are essential for viability, and their gene disruption leads to a phenotype indistinguishable from that observed in cut3-477 and cut14-208, known mutations in SMC4 and SMC2-like subunits. Condensin subunits tagged with GFP were observed to alter dramatically their localization during the cell cycle: they are enriched in the nucleus during mitosis, but cytoplasmic during other stages. This stage-specific alteration in localization requires mitosis-specific phosphorylation of the T19 Cdc2 site in Cut3. The T19 site is phosphorylated in vitro by Cdc2 kinase and shows the maximal phosphorylation in metaphase in vivo. Its alanine substitution mutant fails to suppress the temperature-sensitive phenotype of cut3-477, and shows deficiency in condensation, probably because Cut3 T19A remains cytoplasmic. Therefore, direct Cdc2 phosphorylation of fission yeast condensin may facilitate its nuclear accumulation during mitosis (Satani, 2000).
The characterization of a human condensin complex purified from HeLa cell nuclear extracts is reported. The human 13S complex has exactly the same composition as its Xenopus counterpart, being composed of two structural maintenance of chromosomes (human chromosome-associated polypeptide [hCAP-C and hCAP-E) subunits and three non-structural maintenance of chromosomes (hCAP-D2/CNAP1, hCAP-G, and hCAP-H/BRRN) subunits. Human condensin purified from asynchronous HeLa cell cultures fails to reconfigure DNA structure in vitro. When phosphorylated by purified cdc2-cyclin B, however, it gains the ability to introduce positive supercoils into DNA in the presence of ATP and topoisomerase I. Strikingly, human condensin can induce chromosome condensation when added back into a Xenopus egg extract that has been immunodepleted of endogenous condensin. Thus, the structure and function of the condensin complex are both highly conserved between Xenopus and humans, underscoring condensin's fundamental importance in mitotic chromosome dynamics in eukaryotic cells (Kimura, 2001).
Five genes encoding condensin components in Saccharomyces cerevisiae have been characterized. All genes are essential for cell viability and encode proteins that form a complex in vivo. New mutant alleles of the genes encoding the core subunits of this complex, smc2-8 and smc4-1, have been characterized. Both SMC2 and SMC4 are essential for chromosome transmission in anaphase. Mutations in these genes cause defects in establishing condensation of unique (chromosome VIII arm) and repetitive (rDNA) regions of the genome but do not impair sister chromatid cohesion. In vivo localization of Smc4p fused to green fluorescent protein shows that, unexpectedly, in S. cerevisiae the condensin complex concentrates in the rDNA region at the G2/M phase of the cell cycle. rDNA segregation in mitosis is delayed and/or stalled in smc2 and smc4 mutants, compared with separation of pericentromeric and distal arm regions. Mitotic transmission of chromosome III carrying the rDNA translocation is impaired in smc2 and smc4 mutants. Thus, the condensin complex in S. cerevisiae has a specialized function in mitotic segregation of the rDNA locus. Chromatin immunoprecipitation (ChIP) analysis reveals that condensin is physically associated with rDNA in vivo. Thus, the rDNA array is the first identified set of DNA sequences specifically bound by condensin in vivo (Freeman, 2000).
Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Two human SMC family proteins, hCAP-C and hCAP-E, form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E has been determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3 (Schmiesing, 2000).
In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of the Xenopus condensin complex. To better understand the role of Barren in vivo, conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae were generated. Barren is an essential protein required for chromosome condensation in vivo and it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. However, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics (Lavoie, 2000).
This work describes BRN1, the budding yeast homolog of Drosophila Barren and Xenopus condensin subunit XCAP-H. The Drosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation. Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase. Chromatid cohesion before anaphase is properly maintained in the mutants. Some brn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle. Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis. Temperature-sensitive mutations of BRN1 can be suppressed by overexpression of a novel gene YCG1, which is homologous to another Xenopus condensin subunit, XCAP-G. Overexpression of SMC2, a gene necessary for chromosome condensation, and a homolog of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex (Ouspenski, 2000).
Sister chromatid separation is initiated at anaphase onset by the activation of separase, which removes cohesins from chromosomes. However, it remains elusive how sister chromatid separation is completed along the entire chromosome length. This study found that, during early anaphase in Saccharomyces cerevisiae, sister chromatids separate gradually from centromeres to telomeres, accompanied by regional chromosome stretching and subsequent recoiling. The stretching results from residual cohesion between sister chromatids, which prevents their immediate separation. This residual cohesion is at least partly dependent on cohesins that have escaped removal by separase at anaphase onset. Meanwhile, recoiling of a stretched chromosome region requires condensins and generates forces to remove residual cohesion. Evidence is provided that condensins promote chromosome recoiling directly in vivo, which is distinct from their known function in resolving sister chromatids. This work identifies residual sister chromatid cohesion during early anaphase and reveals condensins' roles in chromosome recoiling, which eliminates residual cohesion to complete sister chromatid separation (Renshaw, 2010).
Successful segregation of chromosomes during mitosis and meiosis depends on the action of the ring-shaped condensin complex, but how condensin ensures the complete disjunction of sister chromatids is unknown. This study shows that the failure to segregate chromosome arms, which results from condensin release from chromosomes by proteolytic cleavage of its ring structure, leads to a DNA damage checkpoint-dependent cell-cycle arrest. Checkpoint activation is triggered by the formation of chromosome breaks during cytokinesis, which proceeds with normal timing despite the presence of lagging chromosome arms. Remarkably, enforcing condensin ring reclosure by chemically induced dimerization just before entry into anaphase is sufficient to restore chromosome arm segregation. It is suggested that topological entrapment of chromosome arms by condensin rings ensures their clearance from the cleavage plane and thereby avoids their breakage during cytokinesis (Cuylen, 2013).
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